Abstract

Erythropoietin (EPO) is the principal cytokine regulating erythropoiesis through its receptor, EPOR. Interestingly, EPORs are also found on immune cells with incompletely understood functions. Here, we show that EPO inhibits the induction of proinflammatory genes including tumor necrosis factor (TNF)-α and inducible nitric oxide (NO) synthase in activated macrophages, which is mechanistically attributable to blockage of nuclear factor (NF)-κB p65 activation by EPO. Accordingly, in systemic Salmonella infection, treatment of mice with EPO results in reduced survival and impaired pathogen clearance because of diminished formation of anti-microbial effector molecules such as TNF-α and NO. However, neutralization of endogenous EPO or genetic ablation of Epor promotes Salmonella elimination. In contrast, in chemically induced colitis, EPO-EPOR interaction decreases the production of NF-κB-inducible immune mediators, thus limiting tissue damage and ameliorating disease severity. These immune-modulatory effects of EPO may be of therapeutic relevance in infectious and inflammatory diseases.

Recombinant EPO Inhibits Proinflammatory Immune Responses in Macrophages In Vitro(A) Thioglycolate-elicited primary peritoneal macrophages were pretreated with PBS or EPO 30 min before the addition of LPS or solvent. Supernatants were analyzed for concentrations of nitrite and of cytokines and data from at least 3 independent experiments were compared by means of Kruskal-Wallis test. Values are depicted as lower quartile, median and upper quartile (boxes) with minimum and maximum ranges, and statistical significances between PBS- and EPO-treatment are indicated. TNF-α and IL-6 concentrations in supernatants of solvent-treated control macrophages remained below the reported detection limits of the corresponding ELISA kits. n.d. denotes not detectable.(B) RAW264.7 macrophage-like cells were transiently transfected with a plasmid containing the luciferase gene under the control of the full-length murine Nos2 promoter. Relative luciferase activity is shown. Data were compared and are depicted as in Figure 1A.

Recombinant EPO Impairs NF-κB Activation and Salmonella Elimination In Vitro(A) RAW264.7 macrophage-like cells were treated with EPO, IFN-γ, and LPS for the indicated time periods. NF-κB DNA binding activity in nuclear extracts was evaluated by means of electrophoretic mobility shift assay (EMSA). The specificity of binding was confirmed by cold competition with a 30-fold excess of the same unlabeled oligonucleotide (far right lane) with the nuclear extract of PBS-treated cells stimulated with IFN-γ and LPS for 120 min. One of three representative experiments is shown.(B) Nuclear proteins were used for specific quantification of NF-κB p65 binding activity by a commercially available chemi-luminescent transcription factor assay at the indicated time points. Data of three independent experiments are expressed as arbitrary light units and shown as means ± SEM and were compared by means of Kruskal-Wallis test. Asterisks indicate statistically significant differences between LPS-stimulated cells pretreated with either PBS or EPO: p < 0.05.(C) Cytoplasmic proteins from parallel experiments described in the legend for Figure 2B were used for evaluating phosphorylation of JAK2, IκB-α, and of NF-κB p65 by means of western blot as described in Experimental Procedures. Total JAK2, IκB-α, and NF-κB p65 protein, respectively, served as loading controls. One of three representative experiments is shown.(D–F) RAW 264.7 cells were pretreated with PBS or EPO as above and subsequently stimulated with LPS or recombinant murine cytokines (D). Alternatively, RAW cells were treated with EPO and the Toll-like receptor (TLR) ligands LPS, PAM2Cys, PAM3Cys, Poly(I:C), or MALP-2 (E). RAW cells were treated with LPS or TNF-α after preincubation with the specific JAK2 inhibitor AG490 (F). Nuclear proteins were used for specific quantification of NF-κB p65 binding activity by a commercially available chemi-luminescent transcription factor assay after 120 min. Data of five independent experiments are depicted as arbitrary light units and shown as means ± SEM.(G) RAW264.7 cells were transiently transfected with murine Nos2 promoter constructs carrying site-specific mutations in one or both NF-κB binding sites (mut-κBI-Nos2-luc and mut-κBII-Nos2-luc or mut-κBI-mut-κBII-Nos2-luc, respectively). Thereafter, macrophages were stimulated with IFN-γ and LPS after preincubation with EPO or solvent. Luciferase activity was measured in a chemi-luminometer and is shown as arbitrary light units.(H–K) RAW264.7 macrophage-like cells (H) or primary peritoneal macrophages (I-K) were infected with S. typhimurium (S. Tm.) at a MOI of 10. After 1 hr, cells were treated with EPO, IFN-γ, AG490 or solvent and incubated for a total of 24 hr. Thereafter, macrophages were lysed and intramacrophage bacteria were enumerated by plating serial dilutions of cell lysates.(L) Primary peritoneal macrophages from Epor+/+ and Epor−/− mice were treated as above after transient transfection with a NF-κB reporter construct. Data of five independent experiments are depicted.

EPO Administration Impairs Pathogen Clearance in Salmonella Infection In Vivo(A and C) C57BL/6 mice were infected i.p. with 500 CFU of S. typhimurium and treated with PBS or EPO on days 3 and 4 after infection. Livers (A) and spleens (C) were removed and formalin-fixed samples were further processed for HE staining. Whereas PBS-treated Salmonella-infected (S. Tm.) animals had microabscesses in livers (indicated by arrowheads) and preserved splenic organ architecture, EPO-treated Salmonella-infected mice presented with macroabscesses in livers (indicated by an arrow) and scattered inflammatory foci in the spleens due to multiple microabscesses. No signs of thrombo-embolic events were observed in either group. Scale bars represent 200 μm.(B and D) Bacterial loads were determined in livers (B) and spleens (D) on day 5 after infection. Data were combined from three independent experiments with similar results. Values from 11 or 12 mice per group were log-transformed and compared by means of Student's t test. Individual values and means are depicted and statistical significances between PBS- and EPO-treatment are indicated.(E) C57BL/6 mice were infected i.p. with 500 CFU of S. typhimurium and treated with PBS or EPO every second day starting on day 1 (24 hr) after infection (n = 14–16 per group). Time points of EPO (or PBS) applications are indicated by arrows. Survival was monitored during an observation period of 7 days. The cumulative survival was analyzed by the log-rank test: p < 0.001 for the comparison of the two groups.(F) Spleen samples were subjected to RNA preparation and quantitative determination of immune gene expression by RT-PCR. Data from 8–12 samples per group are shown as relative abundance of target gene expression in relation to the house-keeping gene hypoxanthin phospho-ribosyl transferase (Hprt).(G) Spleen samples (n = 8–12 per group) were used for the preparation of nuclear extracts and NF-κB p65 binding activity was measured.

Neutralization of Endogenous EPO Reduces Bacterial Loads and Stimulates Antibacterial Immune Effector Pathways(A and B) C57BL/6 mice were infected i.p. with 500 CFU of S. typhimurium and treated with a neutralizing EPO antibody (α-EPO) or isotype control on days 1 and 2 after infection. Bacterial loads were determined in livers (A) and spleens (B) on day 4 after infection.Spleen samples of PBS-treated controls (n = 4-6 per group) and of Salmonella-infected mice (n = 10 per group) were used to study the expression of immune response genes (C) and NF-κB p65 binding activity (D).(E) For analysis of survival, C57BL/6 mice were infected i.p. with 500 CFU of S. typhimurium and treated with a neutralizing EPO antibody (or isotype control) every other day starting 2 days after infection as indicated by arrows.

EPOR Functionality on Nonerythroid Cells Regulates Immune Response and Outcome in Salmonella Infection(A) Epor+/+ and Epor−/− C57BL/6 mice were infected i.p. with 500 CFU of S. typhimurium and treated with PBS or EPO (5 U/g body weight) on days 3 and 4 after infection. Liver histology showed macroabscesses (indicated by an arrow) in EPO-treated Epor+/+ mice and micro-abscesses (indicated by arrowheads) in animals assigned to the other three treatment groups (A). Scale bars represent 200 μm.(B and C) Bacterial loads were determined in livers (B) and spleens (C) on day 5 after infection.(D) Peritoneal macrophages of these mice were seeded in gentamicin-containing RPMI and intracellular bacterial loads were evaluated after 1 hr. Data were compared by Kruskal-Wallis test and statistical significances are indicated.(E and F) Spleen samples of these mice (n = 6–12 per group) were used to measure the expression of immune response genes (E) and NF-κB p65 binding activity (F). Statistical significant differences between Epor+/+ treated with either PBS or EPO and between Epor+/+ and Epor−/− treated with PBS are indicated.(G) For comparison of survival, Epor+/+ and Epor−/− C57BL/6 mice were infected i.p. with 500 CFU of S. typhimurium and treated with EPO at a dose of 5 U/g body weight (or PBS) every other day starting 1 day after infection as indicated by arrows. The cumulative survival was analyzed by the log-rank test: p < 0.001 for the comparison of Epor+/+ treated mice with either PBS or EPO; p < 0.001 for the comparison of Epor+/+ and Epor−/− mice treated with PBS.

EPO Treatment Downregulates Proinflammatory Immune Pathways and Improves Disease Activity in TNBS-Induced Colitis(A) SJL/J mice were subjected to cutaneous immunization of TNBS diluted in EtOH and then to intrarectal administration of TNBS diluted in EtOH; control mice were treated with PBS diluted in EtOH. Subsequently, mice were injected with EPO (or PBS) on days 2, 3, and 4 after induction of colitis as indicated by arrows. The change in weight is expressed as percentage of body weight from day 0, and data are shown as means ± SEM for 10 mice per group.(B) Histopathological colitis scores for EtOH-instilled and TNBS-treated mice described in the legend to Figure 5A (n = 10 per group). Each point represents an individual mouse. Values are depicted as lower quartile, median, and upper quartile (boxes) with minimum and maximum ranges, and statistically significant differences between means of TNBS mice treated with either PBS or EPO are indicated.(C and D) Photomicrographs of HE-stained colonic sections showed mucosal thickening, epithelial hyperplasia and inflammation (arrow) in PBS-treated TNBS-mice (C) and nearly unaffected mucosa (arrow head) observed in EPO-treated TNBS-mice (D). Colon histology is shown at low (left) and high (right) magnification. Scale bars: 200 μm (left) or 100 μm (right).(E and F) qRT-PCR analysis of immune response genes (E) and NF-κB p65 binding activity (F) in colons of these mice (n = 10 per group).

EPO Treatment Downregulates Proinflammatory Immune Pathways and Improves Disease Activity in DSS-Induced ColitisEpor−/− mice and Epor+/+ littermates on a C57BL/6 background were administered 3% DSS dissolved in water or water alone (controls) for 7 consecutive days. Thereafter, DSS was replaced by drinking water and all animals were followed up for another 7 days. Subsequently, mice were injected with EPO (or PBS) on days 7, 8, and 9 after induction of colitis as indicated by arrows.(A) Changes in body weight as combined from two independent experiments and 5–14 DSS-treated mice per group are presented and were compared as detailed in the legend to Figure 6. Data of mice receiving drinking water are not depicted. Statistical significant differences between DSS-treated Epor+/+ mice receiving either PBS or EPO are indicated.(B) Histopathological colitis scores for mice administered either water or DSS (n = 5–14 per group) with each point representing an individual mouse.(C and D) qRT-PCR analysis of immune response genes (C) and NF-κB p65 binding activity (D) in colons of these mice (n = 5–14 per group).